Healthy synapse function relies on precise structural alignment between the presynaptic active zone (AZ) and the postsynaptic density (PSD). A central question is whether localization of vesicle fusion sites influences the efficacy of synapti transmission. The question is important because receptor activation at glutamatergic synapses is limited by both biophysical properties of the receptors themselves and their distance from vesicle release sites. Thus, release events can fail to activate all synaptic receptors. Recently, our lab found that PSD scaffolding proteins cluster receptors into nanometer-scale subregions. This organization is expected to increase the impact of fusion site organization: simulations show that EPSC amplitude at synapses with clustered receptor distributions is greatest when vesicle fusion is aligned with postsynaptic clusters. However, it has not been previously possible to precisely localize vesicle fusion sites, and so there is little information about where within te AZ vesicles fuse. The significance of this issue is emphasized by the growing list of diseases in which cognitive and behavioral deficits appear to stem from disruption of synapse function caused by mutations of genes such as RIM1 and Munc13 that encode synaptic proteins. To map vesicle fusion sites, I developed a novel technique to localize single-vesicle fusion with high spatial resolution, which I call pHluorin uncovering sites of exocytosis or pHuse. Using this approach, I mapped the pattern of evoked or spontaneous vesicle fusion at individual presynaptic terminals of cultured hippocampal neurons. Spontaneous release of neurotransmitter was previously thought to be biological noise but has recently been linked to distinct physiological functions. Interestingly, there is controversy over whether spontaneous and evoked release utilize different vesicle pools, involve different trafficking and fusion machinery, or activate different groups of receptors. All these factors suggest that spontaneous and evoked fusion could take place at spatially distinct regions of the AZ, but this has not been tested. My preliminary data using pHuse indicate that evoked and spontaneous fusion in fact occur over different subregions of the terminal. I hypothesize that the spatial distributions of evoked and spontaneous vesicle fusion are differentially regulated by specific active zone proteins and activity. Mouse mutants suggest that two key presynaptic proteins may mediate this effect: loss of the dominant RIM isoform RIM1a specifically disrupts evoked but not spontaneous release57 while loss of Munc13 disrupts both58. Thus, I will test whether these proteins maintain the differential distribution of evoked and spontaneous release sites. Furthermore, molecular remodeling of the AZ to alter vesicle release has been proposed to underlie changes in presynaptic function associated with synaptic plasticity. Thus, I will assess whether the spatial distribution of evoked and spontaneous fusion within the AZ is differentially regulated by key paradigms of synaptic plasticity. In sum, these Aims will help elucidate the organization and regulation of a key aspect of synaptic function, and test an unexpected new mechanism of activity-driven synaptic remodeling.